Microarray based sample dectection system

ABSTRACT

A microarray assembly for detection of a target molecule is disclosed. The microarray assemblies comprise an array chamber having a microarray located therein and features that facilitate liquid movement within the array chamber. Also disclosed are methods for making the microarray assembly using rollable films and methods for detecting microarray spots using an internal control fluorophore in the array spot.

This application is a continuation of U.S. application Ser. No.15/280,654, filed on Sep. 29, 2016, which is a divisional of U.S.application Ser. No. 14/294,683, filed on Jun. 3, 2014, now U.S. Pat.No. 9,493,815, which is a continuation of U.S. application Ser. No.13/446,291, filed on Apr. 13, 2012, now U.S. Pat. No. 8,828,912, andclaims the priority of U.S. Provisional Patent Application No.61/475,107, filed on Apr. 13, 2011. This application also claimspriority to U.S. continuation-in-part application Ser. No. 12/886,201,filed on Sep. 20, 2010, which claims priority to U.S. ProvisionalApplication No. 61/272,397, filed on Sep. 21, 2009. The entirety of theaforementioned applications is incorporated herein by reference.

FIELD

The technical field is microfluidic systems and, in particular,microfluidic systems having a microarray for sample detection.

BACKGROUND

Microarrays are most prevalent in research laboratories as tools forprofiling gene expression levels because thousands of probes caninterrogate a single sample. Their utility is less ubiquitous asdiagnostics for clinical, environmental, and agricultural applicationsdespite their information density, redundancy, embedded controls(positive, negative), and analytical sensitivity. The barrier toadoption of microarrays as diagnostics tests is predominantly due totheir operational complexity and cost (often hundreds of dollars pertest), as well as technical problems associated with microfluidicdevices containing a microarray, such as the unpredictable behavior offluid flow caused by air bubbles in the microfluidic devices. Forexample, bubbles can clog channels, interfere with biochemical reactions(particularly those that require surface interactions), cause improperproportioning, interfere with optical reads, and result in unpredictableflow. Unpredictable flow is particularly a problem for systems that relyon steady diffusion of an analyte to a binding partner, such as anoligonucleotide or a capturing antibody. Accordingly, there still existsa need for microarray-based microfluidic detection systems that aredesigned to provide predictable fluid flow and can be manufactured at alow cost.

SUMMARY

One aspect of the present application relates to a microarray assemblyfor detection of a target molecule in a sample. In one embodiment, themicroarray assembly comprises: an array chamber with a sample inlet at afirst end, a sample outlet at a second end, a top interior surface, abottom interior surface, side walls and a microarray located on thebottom interior surface; and a waste chamber that is in fluidcommunication with the outlet of the array chamber, wherein the arraychamber comprises a hydrophilic interior surface positioned tofacilitate complete filling of the array chamber by a water-based fluidand the continuous flow of the fluid from the sample inlet to the sampleoutlet and wherein the cross-sectional area at the first end of thearray chamber is larger than the cross-sectional area at the second endof the array chamber.

In another embodiment, the microarray assembly comprises: an arraychamber with a sample inlet, a sample outlet, a top interior surface, abottom interior surface, side walls and a microarray located on thebottom interior surface; a waste chamber comprising a waste inlet and anabsorbent material; and a channel having an expansion section with afirst end proximate to the outlet of the array chamber and a second endproximate to the inlet of the waste chamber, wherein the top interiorsurface is a hydrophilic surface that facilitates complete filling ofthe array chamber by an aqueous fluid and wherein the cross-sectionalarea at the first end of the expansion section is smaller than thecross-sectional area at the second end of the expansion section.

In another embodiment, the microarray assembly comprises: an arraychamber with a sample inlet at a first end, a sample outlet at a secondend, a top interior surface, a bottom interior surface, side walls and amicroarray located on the bottom surface; and a waste chamber that is influid communication with the outlet of the array chamber, wherein thearray chamber comprises a hydrophilic interior surface positioned tofacilitate complete filling of the array chamber by an aqueous-basedfluid and channels with rectangular cross-sectional areas patterned ontothe bottom interior surface and/or the top interior surfaces to promotedrying.

Another aspect of the present application relates to a method forcontrolling the quality of manufacturing array elements in a microarray.The method comprises the steps of illuminating a microarray having aplurality of array spots with light waves to produce fluorescence fromeach array spot; measuring fluorescence intensity for each array spotwherein the fluorescence is produced by an internal quality controlfluorophore; producing a fluorescent image of the microarray;determining information for each array spot based on the fluorescentimage; and encoding the information in a barcode, memory device or RFIDtag, wherein the barcode, memory device or RFID tag is associated withthe microarray.

Another aspect of the present application relates to a method for makinga microarray assembly. The method comprises the steps of unrolling asubstrate film by one or more substrate film reels; printing microarraysonto the unrolled substrate film; laminating a spacer film on top of theprinted substrate film, wherein the spacer film is pre-cut to providespace for an array chamber prior to the placing step and is placed ontop of the printed substrate film by one or more spacer film reels;laminating a cover film on top of the spacer film to form a layeredmicroarray structure; and cutting the layered microarray structure intoindividual microarray assemblies.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description will refer to the following drawings:

FIG. 1A is a schematic of an embodiment of a microarray assembly thatcontains a reservoir, a decreasing cross-sectional area array chamber,an array of spots, a waste chamber, and an absorbent. FIG. 1B is across-sectional view of the array assembly in FIG. 1A.

FIG. 2 is a close-up view of the array chamber showing a linear array ofspots printed at the bottom of the chamber that has a decreasingcross-sectional area.

FIG. 3 is a microarray assembly with an expanding channel connecting thearray chamber to the waste chamber.

FIG. 4A is a schematic showing an array chamber with small rectangularchannels that are perpendicular to the direction of the liquid flowinside the chamber. FIG. 4B is a schematic showing an array chamber withsmall rectangular channels that are parallel to the direction of theliquid flow inside the reaction chamber. FIG. 4C is a schematic showingan array chamber with small rectangular channels that are perpendicularor parallel to the direction of the liquid flow within the reactionchamber. FIG. 4D is a schematic showing an array chamber with smallrectangular channels that form an angle to the direction of the liquidflow within the reaction chamber.

FIG. 5 shows a schematic of a continuous assembly line for manufacturinglab-on-a-film devices.

FIG. 6 shows an array map with a serial dilution of Cy5 and Cy3 spots.

FIG. 7 shows an image of a blunt-pin print head.

FIG. 8 shows bright field images of arrays printed on a polyester thinfilm with the vacuum manifold before polymerization and afterpolymerization, as well as a fluorescence image of a Cy3 array.

FIG. 9 shows a picture of a thin-film vacuum manifold for blunt pinprinting.

FIG. 10 shows a fluorescence image following PCR of materials that wereassembled with rollable materials including a polyester film that thearray was printed on.

FIG. 11 is a composite of pictures showing a reel-to-reel printing setupwith a BioDot Ultranon-contact array printer (top panel) and videoframes of non-contact printing using the BioDot Ultra on a moving filmthat has not been chemically treated or modified (bottom panels).

FIG. 12 shows a red channel fluorescence image of the MRSA arraycaptured during factory QC to extract spot parameters.

FIG. 13 shows a green channel fluorescence image of hybridized arrayimaged by end-user's imager. The imager software utilized the array QCdata to place the grid and circles around each individual spot.

FIG. 14 shows a fluorescence image of hybridized array imaged byend-user equipment without the use of QC data, making it morechallenging to place the grid and circles around each individual spot.

DETAILED DESCRIPTION

This description is intended to be read in connection with theaccompanying drawings, which are to be considered part of the entirewritten description of this invention. The drawing figures are notnecessarily to scale and certain features of the invention may be shownexaggerated in scale or in somewhat schematic form in the interest ofclarity and conciseness. In the description, relative terms such as“front,” “back” “up,” “down,” “top” and “bottom,” as well as derivativesthereof, should be construed to refer to the orientation as thendescribed or as shown in the drawing figure under discussion. Theserelative terms are for convenience of description and normally are notintended to require a particular orientation. Terms concerningattachments, coupling and the like, such as “connected” and “attached,”refer to a relationship wherein structures are secured or attached toone another either directly or indirectly through interveningstructures, as well as both movable or rigid attachments orrelationships, unless expressly described otherwise.

The term “microarray,” as used herein, refers to an ordered array ofspots presented for binding to ligands of interest. A microarrayconsists of at least two spots. The ligands of interest include, but arenot limited to, nucleic acids (e.g., molecular beacons, aptamers, lockednucleic acids, peptide nucleic acids), proteins, peptides,polysaccharides, antibodies, antigens, viruses, and bacteria.

The term “hydrophilic surface” as used herein, refers to a surface thatwould form a contact angle of 45° or smaller with a drop of pure waterresting on such a surface. The term “hydrophobic surface” as usedherein, refers to a surface that would form a contact angle greater than45° with a drop of pure water resting on such a surface. Contact anglescan be measured using a contact angle goniometer.

The term “array chamber,” as used herein, refers to an enclosed spacearound a microarray that has fluid communication with an inlet and anoutlet either directly or indirectly. The array chamber, when filledwith a liquid sample, allows the microarray to be submerged in theliquid sample so that target molecules in the liquid sample can maintainintimate contact with the microarray probes.

Microarray System Designed to Facilitate Fluid Flow Within the System

One aspect of the present application relates to a microarray-baseddetection system comprising a microarray assembly comprising an arraychamber with a sample inlet, a sample outlet and a microarray locatedtherein, and a waste chamber that is in fluid communication with thearray chamber. The array chamber has a hydrophilic surface positioned tofacilitate complete filling of the array chamber and the fluid flow fromthe array chamber to the waste chamber. The hydrophilic surface contactsa liquid as it enters the array chamber from the sample inlet and allowscomplete filling of the array chamber. In certain embodiments, the arraychamber is in the shape of an elongated channel of variable width and isdirectly connected to the waste chamber. In other embodiments, the arraychamber is connected to the waste chamber through a waste channel.

Surface tension of a liquid sample or a reaction mixture often preventthe liquid sample or reaction mixture from completely filling a smallspace, such as the array chamber of a microarray system. Surface tensionis the result of the attraction between the molecules of the liquidsample by various intermolecular forces. In the bulk of the liquidsample, each molecule is pulled equally in all directions by neighboringliquid molecules, resulting in a net force of zero. At the surface ofthe liquid sample, the molecules are pulled inwards by other moleculesdeeper inside the liquid and are not attracted as intensely by themolecules in the neighboring medium (be it vacuum, air or anotherfluid). Therefore all of the molecules at the surface are subject to aninward force of molecular attraction which can be balanced only by theresistance of the liquid sample to compression. This inward pull tendsto diminish the surface area, and in this respect a liquid surfaceresembles a stretched elastic membrane. Accordingly, the liquid squeezesitself together until it has the locally lowest surface area possible.The net result is that the liquid sample may maintain a near-sphericalshape inside the small space and does not fill the corners, especiallysquare corners of the small space. The typical small gap that separatesthe cover from the microarray surface in an array chamber oftencompresses the liquid into a cylindrical shape.

In the case of microarray systems, the liquid that fills the arraychamber is most likely an aqueous solution, such as a hybridizationbuffer or washing buffer. The surface tension of the aqueous solutionmay be overcome by coating at least a portion of the interior surface ofthe array chamber with a hydrophilic material. In some embodiments, themicroarray is located on the bottom surface of the array chamber and thetop surface, or at least a portion of the top surface, of the arraychamber is coated with a hydrophilic coating.

Examples of the hydrophilic material include, but are not limited to,hydrophilic polymers such as polyethylene glycols, polyhydroxyethylmethacrylates, Bionite, poly(N-vinyl lactams), poly(vinylpyrrolidone),poly(ethylene oxide), poly(propylene oxide), polyacrylamides,cellulosics, methyl cellulose, polyanhydrides, polyacrylic acids,polyvinyl alcohols, polyvinyl ethers, alkylphenol ethoxylates, complexpolyol mono-esters, polyoxyethylene esters of oleic acid,polyoxyethylene sorbitan esters of oleic acid, and sorbitan esters offatty acids; inorganic hydrophilic materials such as inorganic oxide,gold, zeolite, and diamond-like carbon; and surfactants such as TritonX-100, Tween, Sodium dodecyl sulfate (SDS), ammonium lauryl sulfate,alkyl sulfate salts, sodium lauryl ether sulfate (SLES), alkyl benzenesulfonate, soaps, fatty acid salts, cetyl trimethylammonium bromide(CTAB) a.k.a. hexadecyl trimethyl ammonium bromide,alkyltrimethylammonium salts, cetylpyridinium chloride (CPC),polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC),benzethonium chloride (BZT), dodecyl betaine, dodecyl dimethylamineoxide, cocamidopropyl betaine, coco ampho glycinate alkyl poly(ethyleneoxide), copolymers of poly(ethylene oxide) and poly(propylene oxide)(commercially called Poloxamers or Poloxamines), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, cocamide TEA.

In some embodiments, one or more surfactants are mixed with reactionpolymers such as polyurethanes and epoxies to serve as a hydrophiliccoating. In other embodiments, the top surface or the bottom surface ofthe array chamber is made hydrophilic by surface treatment such asatmospheric plasma treatment, corona treatment or gas corona treatment.

Examples of hydrophilic tape include, but are not limited to, AdhesivesResearch (AR) tape 90128, AR tape 90469, AR tape 90368, AR tape 90119,AR tape 92276, and AR tape 90741 (Adhesives Research, Inc., Glen Rock,Pa.). Examples of hydrophilic film include, but are not limited to,Vistex® and Visguard® films (Film Specialties Inc., Hillsborough, N.J.),and Lexan HPFAF (GE Plastics, Pittsfield, Mass.). Other hydrophilicsurfaces are available from Surmodics, Inc. (Eden Prairie, Minn.),Biocoat Inc. (Horsham, Pa.), Advanced Surface Technology (Billerica,Mass.), and Hydromer, Inc. (Branchburg, N.J.).

In some embodiments, the hydrophilic tape or film has sufficienttransparency to allow optical interrogation of the microarray from thetop of the array chamber.

The microarray can be any type of microarray, including but not limitedto oligonucleotide microarrays and protein microarrays. In oneembodiment, the microarray is an antibody array and the microarraysystem is used for capturing and labeling target antigens. In oneembodiment, the microarray is formed using the printing gel spots methoddescribed in e.g., U.S. Pat. Nos. 5,741,700, 5,770,721, 5,981,734,6,656,725 and U.S. patent application Ser. Nos. 10/068,474, 11/425,667and 60/793,176, all of which are hereby incorporated by reference intheir entirety. In certain embodiments, the microarray comprises aplurality of array spots printed on an array substrate that forms thebottom of the array chamber. In some embodiments, the array substrate isglass or plastic.

In certain embodiments, the array spots contain an internal controlfluorophore having an emission spectrum that is different from those ofthe fluorophores associated with target molecules (i.e., the targetmolecules will be labeled with fluorophores that have emission spectrathat are different from the emission spectrum of the internal controlfluorophore). This internal control may be analyzed in the field orduring manufacturing to improve quality. The internal control wouldprovide a quantitative means of assessing the fluorescence intensity(e.g., average, mean or integral) of the spot, which may vary due todrop diameter, morphology, porosity, or any factor that may change thereproducibility from spot to spot. Factors that influence theseproperties include UV dosage, temperature, surface properties,synthesis, viscosity, condensation, washing (i.e., due to effects causedby differences in temperature, viscosity, flow rate, stringency oranything that may influence the removal or distortion of the spots),depth of pin immersion in the polymer solution for pin printingtechnologies or any property that could influence the morphology of gelelements or concentration of the probes therein. Imaging in the fieldwould additionally account for: misuse by the user, destruction of thegel elements due to poor handling, washing of the gel elements,increased brightness due to the presence of salts, thermocycling, hightemperature conditions decreasing fluorescent yield, low temperaturecondition increasing fluorescent yield, shelf-life degradation, and/oranything that contributes to the change in fluorescence signal followingthe initial QA/QC during manufacture of the arrays.

Examples of fluorophores include, but are not limited to, pyrene,7-methoxycoumarin, cascade blue, 6-MI, 3-MI, 7-aminocoumarin-X (AMCA-X),6-MAP, pacific blue, marina blue, dimethylaminocoumarin, BODIPY 493/503,BODIPY-FI-X, DTAF (5-DTAF), 6-FAM (fluorescein), dansyl-X, Oregon green500, Oregon green 488 (5 isomer), rhodol green, Oregon green 514,rhodamine green-X, NBD-X, TET, 2′4′5′7′-tetrabromosulfonefluorescein,BODIPY-FI BR₂, BODIPY-R6G, 6-JOE, BODIPY 530/550, HEX, carboxyrhodamine6G, BODIPY 558/568, BODIPY-TMR-X, PyMPO, BODIPY 564/570, Cy3, TAMRA-X,Rhodamine Red-X, BODIPY 576/589, BODIPY 581/591, Texas Red-X, Cy3.5,ROX, BODIPY-TR, Syto-81, Cy5, napthofluorescein, Cy5.5, VIC, SYBR greenI, and SYBR green II.

In other embodiments, the internal control is a colorimetric signalchange, which is distinct from spot to spot. In other embodiments, theinternal control is a chemiluminescence signal change, which is distinctfrom spot to spot. In yet other embodiments, the internal control is anelectrochemical signal change, which is distinct from spot to spot.

In certain embodiments, the array spots are gel spots containing a firstfluorophore (e.g., Cy5). The targets in the sample are labeled with asecond fluorophore (e.g., Cy3) during PCR and subsequently hybridize toprobes that are covalently attached to the gel drop polymer. The firstfluorophore has a different emission peak than the second fluorophore.In this setting, the first fluorophore (e.g., Cy5) serves to allow exactlocation of the gel spots with an imaging system that can detect boththe first and the second fluorophores (e.g., Cy3 and Cy5).

In some embodiments, the imaging system is a component of themicroarray-based sample detection system. In other embodiments, theimaging system is part of a machine vision system used duringmanufacturing the microarray assembly such that the coordinates of eachspot can be precisely determined during inspection. These coordinatesare uploaded onto a barcode or RFID tag that is attached to themicroarray assembly for future analysis. For this approach to beeffective, the first fluorophore (i.e., the internal controlfluorophore) coordinates require that the second fluorophore (i.e., thetarget fluorophore) reference fiducials are included as part of theassembly map, so that the grid can be placed. However, unlikeconventional scheme that either attempt to place a grid based onprecisely spaced spots or require two color fluorescence imagers, thedisclosed scheme uses the coordinates from the barcode to place fixedcircles for spot detection. Location of the first fluorophore (i.e., theinternal control fluorophore) spots can be used with a thresholdingalgorithm to find the centers, which are then used for placement offixed circles.

A benefit of the use of machine vision to identify spots is that thesame system can be used to reject spots without rejecting the entiremicroarray, which would increase yield. Spots can be rejected based on anumber of criteria such as internal control fluorescence intensityvalues that are out of bounds, asymmetry, and diameter. Therefore, someembodiments of the present application relate to a method forcontrolling the quality of manufacturing array elements in a microarray,comprising: illuminating a microarray having a plurality of array spotswith light waves to produce fluorescence from each array spot; measuringfluorescence intensity for each array spot wherein the fluorescence isproduced by an internal quality control fluorophore; producing afluorescent image of the microarray; determining information for eacharray spot based on the fluorescent image; and encoding the informationin a barcode, memory device or RFID tag, wherein the barcode, memorydevice or RFID tag is associated with the microarray. The informationfor each array spot may comprise the location of each spot, thefluorescence intensity of each spot, the diameter of each spot and themorphology of each spot. A microarray image analysis may be conducted byplacing fixed circles for each microarray spot on the image of amicroarray using the spot location information determined based theinternal control fluorescence.

In one embodiment, the present application provides a method formicroarray image analysis. The method comprises the steps of obtainingan image of a microarray, placing a fixed spot border circle around eachmicroarray spot on the image of the microarray based on the array spotlocation information obtained through the internal control fluorescencein the array spots as described above; measuring a target fluorescenceintensity within the fixed spot border circle for each array spot, anddetermining the amount of a target molecule in a sample based on theratio of the target fluorescence intensity to the internal fluorescenceintensity at each array spot.

In another embodiment, a method for microarray image analysis includesthe following steps: determining a target fluorescence intensity for atarget spot in a microarray; determining an internal fluorescenceintensity for the target spot in the microarray; determining a signalstrength for the target spot in the microarray, wherein the signalstrength is a ratio of the target fluorescence intensity to the internalfluorescence intensity, wherein the internal fluorescence intensity forthe target spot in the microarray is determined as described earlier.

In another embodiment, the present application provides a method forimaging array elements in a microarray. The method includes the steps ofilluminating a microarray having a plurality of array spot with lightwaves of a first wavelength to produce fluorescence from an internalcontrol fluorophore; determining location of array spots of themicroarray based on fluorescence produced by the internal controlfluorophore (control fluorescence); illuminating the microarray withlight waves of a second wavelength to produce fluorescence from a targetfluorophore that is associated, directly or indirectly, to a targetmolecule that binds to an array spot; measuring fluorescence produced bythe target fluorophore (target fluorescence); and determining the amountof the target molecule in the sample based on the control fluorescenceintensity-to-target fluorescence intensity ratio in relevant arrayspots.

The waste chamber can be of any shape and typically has a volume that isgreater than the volume of the array chamber. In one embodiment, thewaste chamber is formed in a gasket tape which is then attached to thesubstrate on which the microarray is printed. In yet another embodiment,the substrate has a cut-out on its top surface. The cut-out has a sizeand position that match the size and position of the waste chamber inthe gasket so that the waste chamber, once formed between the substrateand the gasket, would have a thickness that is greater than thethickness of the array chamber. In another embodiment, the substrate ismade of a plastic material so that a cut-out may be easily made on thesubstrate. In yet another embodiment, both the array chamber and thewaste chamber are formed in the substrate without using the gasket. Thewaste chamber, however, may have a depth that is greater than the depthof the array chamber.

In one embodiment, the waste chamber contains an absorbent that, once incontact with the liquid in the array chamber, wicks the liquid from thearray chamber, therefore allowing the microarray to be read in a drystate.

The absorbent can be any material capable of retention of a relativelylarge volume of liquid. In one embodiment, the absorbent is made of anaggregate of fibers. In another embodiment, the absorbent is a nonwovenfabric produced in a through-air bonding process. The constituent fibersof the nonwoven fabric can be hydrophilic synthetic fibers, naturalcellulose fibers of pulp or the like, or regenerated cellulose fibers.The fibers may be coated or infiltrated with a surfactant or ahydrophilic oil to improve liquid absorbance. Not limited to thethrough-air bonding process, the nonwoven fabric for use herein may beproduced in any other process such as a spun-bonding process, an airlaying process, a spun-lacing process, etc. In one embodiment, theabsorbent is a cellulose paper (C048) from Millipore (Billerica, Mass.).

In some embodiments, the waste chamber is vented to the atmospherethrough a vent. In one embodiment, the vent is created by simplypunching a hole in the cover of the waste chamber.

In another embodiment, the liquid in the array chamber is removed byforcing the liquid inside the reservoir into the array chamber andestablishing a contact between the liquid in the array chamber and theabsorbent in the waste chamber. The contact may be established byapplying a pressure to the liquid in the array chamber to push theliquid out of the array chamber or by applying suction at a vent of thewaste chamber to pull the liquid out of the array chamber. A pressure tothe liquid in the array chamber may be generated by applying a pressurethrough a check valve (e.g., using a pipette or a syringe). If the arraychamber is covered only with a hydrophilic tape or a hydrophilic film, apressure to the liquid inside the array chamber may be generated bysimply pressing the hydrophilic tape or film that form the top surfaceof the array chamber. Alternatively, the contact between the liquid inthe array chamber and the absorbent may be established by placement ofthe absorbent near the array chamber such that the absorbent touches theliquid inside the channel.

Once a contact is established, the liquid in the array chamber is wickedinto the absorbent in the waste chamber through the array chamber. Theflow rate of the liquid is determined by the size of the array chamber,the surface tension and viscosity of the liquid, and the wicking rate ofthe absorbent. In addition, the flow rate decreases as the absorbentbecomes more saturated.

In another embodiment, the microarray system further contains a one-wayvalve for introducing a liquid (e.g., a sample, a PCR buffer withtarget, a hybridization buffer, or a washing buffer) into the arraychamber. The sample is introduced into the array chamber through theone-way valve to prevent environmental contamination, which is animportant concern in certain applications such as the detection ofbiological warfare agents. The one-way valve can be a check valve, adome valve or a duckbill valve that is placed at the inlet of the arraychamber. Dome valves of various sizes are commercially available e.g.,from Minivalve International (Yellow Springs, Ohio).

In some embodiments, the side walls of the array chamber are hydrophobicto trap bubbles. In other embodiments, the array chamber has ahydrophilic cover that is configured such that a hydrophilic region iscreated near the outlet of the array chamber. In a related embodiment,the hydrophilic region is created with hydrophilic gel elements.

In another embodiment, the inlet of the array chamber contains apierceable membrane/tape or a dome valve, check valve or duckbill valveto allow washing to occur without causing the content inside the arraychamber to be liberated from the microarray assembly.

In another embodiment, the microarray system further contains areservoir for introducing a liquid into the array chamber. In a relatedembodiment, the reservoir is loosely bound to the device so that it canbe snapped off and removed for imaging in conventional microarray orcolorimetric readers. In another embodiment, the array chamber isconnected to multiple waste chambers to ensure that wicking occurs atthe appropriate interval.

In the event that an air bubble is introduced into the array chamber,the air bubble may be lodged in the array chamber and partially orcompletely block liquid flow in the array chamber. The air bubble mayalso stop the wicking action of the absorbent if the air bubble islocated right at the interface of the liquid and the absorbent. In someembodiments, the array chamber of the microarray assembly is shaped tofacilitate bubble movement within the array chamber. In someembodiments, the array chamber has a cross-sectional area that decreasescontinuously, or in a stepwise fashion, from one end of the chamber tothe other end of the chamber so as to facilitate liquid movement, aswell as the bubble movement, from the inlet of the array chamber to theoutlet of the array chamber.

FIG. 1A shows an embodiment of a microarray assembly 100 designed tofacilitate the removal of air bubbles in the array chamber. Themicroarray assembly 100 comprises a funnel-shaped array chamber 110spanning from a sample inlet 112 to an outlet 114, which opens into awaste chamber 120 having an absorbent 122. The microarray chamber 110contains a plurality of microarray spots 130 that are positioned on topof a substrate 150 (see FIG. 1C), which also forms the bottom of thearray chamber 110. In certain embodiments, the array chamber 110 isconnected to a reservoir 140. In this embodiment, the array chamber 110has a progressively decreasing cross-sectional area towards thedirection of the waste chamber 120, thus the capillary pressurecontinuously increases as the liquid in the array chamber 110 approachesthe waste chamber 120. The pressure difference leads to liquid movementtowards the absorbent 122 in the waste chamber 120. In other words, theshape of the array chamber 110 provides continuous wicking of a liquidin the array chamber 110 in the direction of the waste chamber 120 untilthe liquid reaches the absorbent 122 in the waste chamber 120. In someembodiments, the cross section area at the inlet end of the arraychamber 110 is at least 2-times, 3-times, 4-times or 5-times larger thanthe cross section area at the outlet end of the array chamber 110.

In one embodiment, the array chamber 110 has a trapezoid shape withdimensions that range from 0.5 to 20 mm on the inlet end and 0.1 to 5 mmon the outlet end. In another embodiment, the array chamber 110comprises a series of steps that have a progressively smallercross-sectional area from the inlet end to the outlet end. Thesefeatures are designed to have a small radius of curvature on theadvancing front compared to the receding front, so that air bubbles inthe array chamber 110 advance towards the waste chamber 120, preventingthe aforementioned issues associated with bubbles.

FIG. 1B is a cross-sectional view of the microarray assembly 100 alongline AA in FIG. 1A. In this embodiment, the microarray assembly 100comprises the array substrate layer 150, the spacer layer 160 and thecover layer 170. In one embodiment, the spacer layer is a double-sidedtape, such as an inner gasket tape, with a thickness of 0.25 mm(available from 3M, Part No. 9087). In other embodiments, the arraysubstrate layer 150 is injection molded plastic with features thatcreate the walls of the array chamber 110 and a pocket for the wastechamber 120 and there is no spacer layer 160 in these embodiments.

In other embodiments, a hydrophilic film is laminated to a plastic arraysubstrate 150 with heat and/or pressure to form a hydrophilic surface onwhich the microarray is printed. The lamination may be performed withlaser welding or ultrasonic welding.

FIG. 2 provides a close-up view of the funnel shaped array chamber 110of FIG. 1A. As shown in FIG. 2, the decreasing chamber width or the“wedge” shape of the array chamber enables increasing capillary pressureon the side of the waste chamber 120. This configuration allows bubblesto flow through the array chamber and avoids clogging of the arraychamber 110 by air bubbles. This funnel-shaped narrow chamber 110 alsofacilitates the diffusion of the target molecules in a sample to thearray spots 130. In some embodiments, the sample is loaded into thereservoir 140 and continuously flows through the array chamber 110 andinto the waste chamber 120.

In some embodiments, the microarray spots 130 are arranged in the formof multiple strips (e.g., protein strip array) that are perpendicular tothe flow in the array chamber 110 so as to improve interaction betweenthe target molecule in the sample and the array elements. In oneembodiment, a protein array or a protein strip array is printed insidethe array chamber 110. Proteins extracted from a sample are loaded intothe reservoir 140 and flow through the array spot 130 or strip 130 in acontinuous fashion to enter the waste chamber 120.

A person of ordinary skill in the art would understand that themicroarray assembly 100 may have many variations. For example, theentire microarray assembly 100 may be molded in two halves creating aparting line that spans the center line of the reservoir 140, thesubstrate 150 and the waste chamber 120. The parting line may take acontoured path to allow easy access for hydrophilic surface treatment ofthe top side of the array chamber 110, and/or printing the array spots130 on the top surface of the substrate 150. The top half of the arrayassembly may be treated to be hydrophilic such as with a plasmatreatment, a surfactant or any of the techniques described above, andbonded into place using ultrasonic welding, laser welding, snap fitdesign, glue, tape, or any bonding method. In some embodiments, thecover layer 170 is sized to cover only the chamber areas but not thecomplete top surface of the microarray assembly 100.

FIG. 3 shows another embodiment of a microarray assembly 100 designed tofacilitate the removal of air bubbles in the array chamber 110 as wellas maintain the sample within the array chamber 110 during prolongedexposure to extreme temperatures (up to 95° C.). In this embodiment, themicroarray assembly 100 comprises an array chamber 110 having a sampleinlet 112, a sample outlet 114 and a plurality of microarray spots 130positioned on top of the substrate 150, a waste chamber 120 having anabsorbent 122, an inlet 116, and a vent 124 and a channel 118 thatconnects the sample outlet 114 of the array chamber 110 to the inlet 116of the waste chamber 120. In this embodiment, the channel 118 has anexpansion section 118A and a switchback section 118B. The expansionsection 118A has progressively increasing cross-sectional area towardsthe direction of the waste chamber 120, so that air bubbles in the arraychamber 110, once entering the channel 118, are trapped on the sidewalls of the section 118A and do not block fluid flow in the channel118. The expansion section 118A helps to pin the contact line of theliquid on the convex corners of the section during sample expansion whenthe array chamber 110 is exposed to high temperatures. In oneembodiment, the sidewall of channel 118 is hydrophobic to trap bubbles.In some embodiments, the cross-sectional area at the waste chamber endof the channel 118A is at least 2-times, 3-times, 4-times or 5-timeslarger than the cross-sectional area at the array chamber end of thechannel 118A. In some embodiments, the switchback section 118B containstwo turns to form an S-shaped or Z-shaped channel section. In oneembodiment, the two turns are 90° turns.

In other embodiments, the array chamber 110 is fabricated with smallrectangular channels 180 (i.e., channels with rectangularcross-sectional areas) that are perpendicular to the direction of theflow to provide a means of drying the array (see FIG. 4A). Thesechannels 180 have sharp corners that result in small radius of curvatureof the liquid-air interface, and thus provide high capillary pressuresthat advance liquids along the side walls and to the waste chamber 120.In another embodiment, the rectangular channels 180 are parallel to theliquid flow path (see FIG. 4B). In another embodiment, the rectangularchannels 180 are both parallel and perpendicular to the liquid flow path(see FIG. 4C). In another embodiment the rectangular channels 180intersect the liquid flow path at angles that range from 30 to 120degrees (see FIG. 4D). In another embodiment, the top surface of thesubstrate 150 is roughened to provide the same wicking action along thecrevices of the surface. The top surface could also be roughened suchthat there are square microchannels that are parallel, intersect,perpendicular, or some or all of these. The contact angle at the cornersshould be lower than 90 degrees so as to advance the liquid along thesechannels towards the waste chamber (absorbent). This approach is similarto that of the tracheids (square capillaries) in conifer trees thatallow liquid to advance up the length of trees, overcoming the effectsof hydrostatic pressure

Detection of Target Molecules with the Microarray Assembly

Another aspect of the present application relates to a method of usingthe microarray assembly described above to detect a target molecule in asample. The sample can be any biological sample, such as a swab,nasopharyngeal aspirate or whole blood sample. The total nucleic acidsmay be isolated using techniques well-known to a person of ordinaryskill in the art. In one embodiment, the total nucleic acids areisolated with commercially-available nucleic acid isolation reagents orkits, such as the Qiagen reagents. In another embodiment, the totalnucleic acids are isolated with a sample preparation device developed byAkonni Biosystems. The generalized sequence of events for Akonni'ssample preparation methods includes denaturing the sample in a lysisbuffer; continuous perfusion of the lysed sample over the samplepreparation device; washing and eluting the nucleic acids from thesample preparation device.

The isolated nucleic acids are loaded into the microarray system andamplified within the microarray assembly using methods well-known to oneskilled in the art. After amplification, the microarray assembly isincubated for a period of time at a desired temperature (e.g., 10-60 minat 50-65° C.) to allow the amplicons to hybridization to the microarray.After incubation, the microarray system is washed (e.g., with water) andimaged on a microarray reader (e.g., Akonni's portable microarrayreader). In one embodiment, the microarray system is dried prior toimaging. In another embodiment, the drying procedure is accomplishedwith acetone introduction to the array chamber and/or heating the arraychamber. In another embodiment, amplification of the isolated nucleicacids and labeling of the amplification products occur in an asymmetricPCR master mix containing fluorescently labeled “reverse” primers inlarge excess (e.g., 5-20 fold excess) over unlabeled, “forward” primers.This strategy generates predominantly single-stranded targets with asingle label on their 5′ end.

The array test can be performed with many variations. In one embodiment,the amplified product remains in the reaction chamber afterhybridization and there is no washing before imaging of the microarray.In another embodiment, the amplified product remains in the arraychamber, and the array spots are imaged in real-time duringhybridization in order to show growth curves as described by Khodakov etal., 2008. In yet another embodiment, the array chamber supports aseries of incubation and wash steps for multi-step assays such asELISAs. In one embodiment, the incubation step is performed underperiodic or continuous vibration to improve interaction between thearray elements and the target proteins.

Manufacturing of the Microarray Assembly

Another aspect of the instant application relates to a method formanufacturing microarray assemblies having a substrate layer, a spacerlayer and a cover layer using rollable thin film materials andreel-to-reel equipment. Briefly, rollable film materials are used forthe substrate layer, the spacer layer and the cover layer of themicroarray assembly. The films are layered together by unravelingseveral reels on top of one another, creating a sandwich of desiredcomponents, which are cut to size at the end of the manufacturing line.Specifically, a rollable substrate film is advanced onto a manufacturingplatform. Array spots are printed onto the film, forming microarrayswith a fixed interval between arrays. The printed substrate film is thenlaminated with a rollable spacer tape that has been pre-cut with aseparate reel-to-reel manufacturing method to create space for the arraychamber. A rollable cover film is then laminated on top of the spacerfilm to seal off the array chamber. In some embodiments, the rollablespacer tape is pre-cut to create space for the array chamber and one ormore waste chambers. An absorbent is placed into each waste chamberprior to the lamination of the cover film to the spacer film. The virtueof this manufacturing method is that high volume production can be verycost effective because with standard production equipment, assembly ofthe microarray assemblies can be completely automated at very highspeeds.

The substrate film can be any thin film having a surface that has doublebonded carbon atoms. Preferably, the substrate film has a hydrophobicsurface. Examples of the substrate film include, but are not limited to,polyester films, polyester/polycarbonate blend films,polytetrafluoroethylene, polyethylene, polyetherimide, polyether etherketone, and polystyrene. In some embodiments, the substrate film has athickness in the range of 20-200 microns, preferably 50-125 microns.

The spacer film can be any double-sided tape with a desired thickness.In certain embodiments, the spacer film is made from a hydrophobicmaterial and has a thickness in the range of 20-500 micron, preferably100-300 microns. Examples of the spacer film include, but are notlimited to, polyester films, polyester/polycarbonate blend films,polypropylene, polycarbonate, acetal, poly(methyl methyacrylate), 256Mtape from Adchem, and polytetrafluoroethylene. The cover film can be anythin film with a hydrophilic surface. Examples of hydrophilic filminclude, but are not limited to, Vistex® and Visguard® films (FilmSpecialties Inc., Hillsborough, N.J.), and Lexan HPFAF (GE Plastics,Pittsfield, Mass.). Other hydrophilic surfaces are available fromSurmodics, Inc. (Eden Prairie, Minn.), Biocoat Inc. (Horsham, Pa.),Advanced Surface Technology (Billerica, Mass.), and Hydromer, Inc.(Branchburg, N.J.).

In some embodiments, the cover film has a thickness in the range of25-250 microns, preferably 50-150 microns.

In some embodiments, the microarray is a gel spot microarray printedonto the substrate film with a non-contact microarray printer (e.g., apiezoelectric printer) that allows for printing on a moving film. Insome embodiments, the gel spots comprise probes, such as protein probesor nucleotide probes that are covalently cross-linked to the polymerbackbone by UV-induced co-polymerization.

FIG. 5 shows an embodiment of a reel-to-reel assembly line for themanufacturing of the microarray device of the present application.Briefly, a substrate film 510 is laid onto the assembly line 500 by thesubstrate film reel 512. A gel spot printer 514 prints array spots ontothe substrate film 510. Probes in the gel spots are covalentlycross-linked to the polymer backbone by UV illumination. In oneembodiment, the crosslinking is accomplished via a single-step,Argon-atmosphere, UV-induced co-polymerization process in a UV chamber516. In one embodiment, the thin films are held in place using theinherent tension between reels on the system. This improves UVillumination uniformity on the surface of the thin film by keeping thefilms flat in the UV chambers during polymerization. The crosslinkedmicroarray is washed at the wash station 518, dried by air knives 520and examined by the quality control (QC) camera 522. Defective arrayscan be marked by a reject marker 524 and a spacer film 526 is laminatedonto the substrate film 510 by the spacer tape reel 528. The spacer film526 can be pre-cut prior to lamination to create space for an arraychamber and one or more waste chambers. Absorbents 530 are then added tothe waste chambers using, such that size-on precut pieces of absorbentwith an adhesive backing are placed in the open waste chamber via theabsorbent reel 532. A cover film 534 is then laminated on top of thesubstrate/spacer layer structure by the cover film reel 536. Theassembled layer structure is then cut by the guillotine 538 to produceindividual microarray assemblies.

EXAMPLES Example 1 Method for Compensating Microarray PrintingVariations

Gel drop microarrays with Cy3 and Cy5 fluorophores were printed on tenseparate slides according to the following assembly map. The followingsteps are used for printing the microarray: (1) prepare the appropriateCy3/Cy5 oligo mixture and dry it down on a CentriVap, (2) prepare acopolymer solution (monomer+cross-linker+glycerol+buffer), (3) dissolvethe dried oligo in copolymer solution, (4) place solution into a sourceplate, and (5) use the source plate for arrayprinting/polymerization/washing.

Assembly Map 1 2 3 4 5 6 1 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 2 Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 3 Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 4Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 5 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) 6 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 7 Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 8 Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 9Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 10 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) 11 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 12 Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 13 Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 14Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 15 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) 16 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 17 Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 18 Cy3:Cy5(1:1)Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) 7 8 910 10  1 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  2 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  3 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  4 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  5 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  6 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  7 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  8 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)  9 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 10 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 11 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 12 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 13 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 14 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 15 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 16 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 17 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1) 18 Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1) Cy3:Cy5(1:1)Cy3:Cy5(1:1)

A GenePix 4000B with the following settings was used for analysis: 100%Laser power for both colors, gain of 500 for the red channel and gain of375 for the green channel photomultiplier tube voltage setting, 5 μmresolution, and 175 μm diameter circles. Integrated intensities werecalculated for each spot using the GenePix software, and relativestandard deviations (RSD) were calculated for all 198 Cy5 spots, 198 Cy3spots and the ratio of Cy3/Cy5 spots. As seen in Table 1, thecoefficient of variation (CV) is lower for all 10 slides when using aratio of the Cy3/Cy5 integrated intensity compared to the intensity ofthe Cy3 or Cy5 signals, in some cases by a factor as high as 3. Thisdata support the implementation of an internal fluorescence control,such as a Cy5 dye, that is scanned or imaged as part of themanufacturing QC to compensate for variability due to UV dosage,temperature, surface properties, synthesis, viscosity, condensation,washing (i.e., due to effects caused by differences in temperature,viscosity, flow rate, stringency or anything that may influence theremoval or distortion of the spots), depth of pin immersion in thepolymer solution for pin printing technologies or any property thatcould influence the morphology and or concentration of the probes withina given spot.

TABLE 1 Slide Cy5 RSD Cy3 RSD Cy3/Cy5 RSD 1 17.5% 12.1% 7.6% 2 14.3%10.0% 5.5% 3 10.8% 8.5% 3.1% 4 5.5% 3.7% 3.0% 5 7.5% 6.0% 2.5% 6 5.1%5.0% 1.4% 7 8.5% 6.0% 4.1% 8 11.7% 7.8% 5.1% 9 6.6% 5.0% 3.6% 10 4.6%4.5% 3.5%

Example 2 Method of Image Analysis

The internal fluorescence control has been implemented on Akonni's MRSAmicroarrays and shown to be effective in compensating for thevariability in the intensity of fluorescence. Table 2 shows thefluorescence data of one set of 4 gel drops in MRSA microarrays dopedwith Cy5 fluorophores and MecA probes. Integral signal intensities weretabulated for all 4 replicate drops taken during factory QC(red-channel) and post-hybridization (green-channel). Due to physicaldamage to replicate 3, both the red-channel and green-channel showedsignificantly reduced integral signal intensities for replicate 3. Asthe result of the reduced replicate 3, signal intensity, the relativedeviation is 23.8% and 29.5% for red-channel and green-channels,respectively. When the green-channel and red-channel data is calculatedas a ratio, the relative deviation is reduced to 12.2%. Thisdemonstrates that internal fluorescence control data (red-channel) canbe used to reduce the variability of the microarray image and/ormicroarray production.

TABLE 2 Integral of Signal Intensities Standard Replicate 1 Replicate 2Replicate 3 Replicate 4 Mean Deviation % RSD Red-Channel 1031897 1095959613676 1063218 951187 226522 23.8% Green- 2812769 3707689 19095223874995 3076244 906896 29.5% Channel Green/Red 2.725823 3.3830543.111613 3.644591 3.21627 0.392754 12.2% Ratio

Example 3 Algorithms for Image Generation Algorithm 1

This algorithm takes a pre-hybridization Cy5 QC image of the array andgenerates a data file containing QC parameters of the array.

-   1. Read the Cy5 QC image and create two local copies, one is the    un-altered original (CY5_Original), and another one will be    transformed into a binary image (Cy5_Processed) in steps 2 and 3.-   2. Take the Cy5_Processed image, apply digital filtering and pixel    operation to produce an image with uniform and zero-valued    background.-   3. Threshold the image into a binary image and save as    Cy5_Processed.-   4. Apply particle analysis to the binary image (Cy5_Processed) to    identified, filtered objects based on size. Measure and record    parameters of the objects: center of mass, bounding box, particle    area and ellipticity.-   5. Check to see if the number of objects identified in step 4 meets    minimum requirement, otherwise reject the slide.-   6. Find grid.    -   a. Select one object and assume its center of mass is the grid        origin.    -   b. Form the grid and calculate the pixel location of each grid        cell.    -   c. Apply all objects to the grid and check if at least 80% of        the grid cell that should contain a Cy3 drop, has objects        inside. If yes, the grid has been found and proceed to step 7.        If not, repeat 6A through 6C with a different object's center of        mass as the grid origin.-   7. Rotate the image so the angle formed by the Cy3 drops is less    than 0.2 degree from the horizontal axis.-   8. Fine-tune the grid. Because in step 6, grid origin is determined    by center of mass from an object in the binary image, the center of    mass could deviate slightly from the true center of the object.-   a. Move the grid origin by (0,1), i.e., subtract X-coordinate by 0    pixel and Y-coordinate by 1 pixel.-   b. For each Cy3 drop, calculate the following:    -   i. Deviation X: Distance in X coordinate between center of Cy3        drop and center of its grid cell.    -   ii. Deviation Y: Distance in Y coordinates between center of Cy3        drop and center of its grid cell.-   c. Summarize the deviations for all Cy3 drops, using    score=Sum(abs(DeviationX)+abs(DeviationY)). A lower score means    better grid placement.-   d. Repeat 8A through 8C for a total of 24 combinations, shown in the    table below.-   e.

−2, 2 −1, 2 0, 2  1, 2 2, 2 −2, 1 −1, 1 0, 1  1, 1 2, 1 −2, 0 −1, 0 −1,0  2, 0  −2, −1  −1, −1 0, −1  1, −1  2, −1  −2, −2  −1, −2 0, −2  1, −2 2, −2

-   f. Choose the grid center so its score is the lowest.-   9. Calculate QC data of each spot.    -   a. Deviation X: X coordinate of center of drop MINUS X        coordinate of center of grid cell.    -   b. Deviation Y: Y coordinate of center of drop MINUS Y        coordinate of center of grid cell.    -   c. Reject Flag: Reject a spot based on diameter, ellipticity,        etc.    -   d. Spot Intensity    -   e. Diameter-   10. Write QC data to a text file, refer to Table 3.

TABLE 3 Example Array QC Data. Grid Grid Spot Spot Row Column TypeReject? Grid Center X Grid Center Y Deviation X Deviation Y IntensityDiameter 1 1 Cy3 FALSE 105 88 3 1 2143818 8.85 1 2 Empty FALSE 135 88 00 261 0.00 1 3 Empty FALSE 166 88 0 0 568 0.00 1 4 Empty FALSE 196 88 00 −87 0.00 1 5 Empty FALSE 226 88 0 0 228 0.00 1 6 Cy3 FALSE 257 88 0 31612600 9.72 1 7 Cy3 FALSE 287 88 1 3 1567664 9.02 1 8 Empty FALSE 31888 0 0 506 0.00 1 9 Empty FALSE 348 88 0 0 1426 0.00 1 10 Empty FALSE378 88 0 0 3420 0.00 1 11 Empty FALSE 409 88 0 0 991 0.00 1 12 Cy3 FALSE439 88 −1 3 2216029 9.65 2 1 Empty FALSE 105 119 0 0 −319 0.00 2 2 EmptyFALSE 135 119 0 0 334 0.00 2 3 Probe 31 FALSE 166 119 5 0 1618379 10.122 4 Empty FALSE 196 119 0 0 486 0.00 2 5 Empty FALSE 226 119 0 0 83 0.002 6 H FALSE 257 119 −1 −1 1750396 8.99 2 7 Empty FALSE 287 119 0 0 407090.00 2 8 Empty FALSE 318 119 0 0 −162 0.00 2 9 Probe 31 FALSE 348 119 03 2061064 10.61 2 10 Empty FALSE 378 119 0 0 127 0.00 2 11 Empty FALSE409 119 0 0 289 0.00 2 12 H FALSE 439 119 −1 3 2030635 9.19 3 1 EmptyFALSE 105 149 0 0 1143 0.00 3 2 Probe 14 FALSE 135 149 4 1 2222565 9.433 3 Probe 35 FALSE 166 149 2 1 2088478 9.16 3 4 Empty FALSE 196 149 0 03938 0.00 3 5 Empty FALSE 226 149 0 0 −96 0.00 3 6 Empty FALSE 257 149 00 −33 0.00 3 7 Empty FALSE 287 149 0 0 582 0.00 3 8 Probe 14 FALSE 318149 0 1 2073261 9.90 3 9 Probe 35 FALSE 348 149 2 1 1651170 8.86 3 10Empty FALSE 378 149 0 0 837 0.00 3 11 Empty FALSE 409 149 0 0 370 0.00 312 Empty FALSE 439 149 0 0 315 0.00 4 1 Empty FALSE 105 180 0 0 162 0.004 2 Empty FALSE 135 180 0 0 −179 0.00 4 3 Probe 36 FALSE 166 180 3 21782715 8.49 4 4 Empty FALSE 196 180 0 0 633 0.00 4 5 Probe 29 FALSE 226180 3 0 1715205 9.51 4 6 Empty FALSE 257 180 0 0 274 0.00 4 7 EmptyFALSE 287 180 0 0 242 0.00 4 8 Empty FALSE 318 180 0 0 329 0.00 4 9Probe 36 FALSE 348 180 −1 −1 1666545 9.66 4 10 Empty FALSE 378 180 0 012157 0.00 4 11 Probe 29 FALSE 409 180 −1 1 1706590 10.47 4 12 EmptyFALSE 439 180 0 0 1180 0.00 5 1 Empty FALSE 105 210 0 0 308 0.00 5 2Empty FALSE 135 210 0 0 180 0.00 5 3 Probe 37 FALSE 166 210 2 1 15374559.70 5 4 dN20 FALSE 196 210 1 0 1854849 10.35 5 5 Empty FALSE 226 210 00 486 0.00 5 6 Probe 90 FALSE 257 210 2 1 1697651 9.01 5 7 Empty FALSE287 210 0 0 −115 0.00 5 8 Empty FALSE 318 210 0 0 −382 0.00 5 9 Probe 37FALSE 348 210 1 2 2009715 9.84 5 10 dN20 FALSE 378 210 0 1 2187695 10.805 11 Empty FALSE 409 210 0 0 1099 0.00 5 12 Probe 90 FALSE 439 210 0 02007504 10.03 6 1 Cy3 FALSE 105 240 5 0 1264671 8.35 6 2 Empty FALSE 135240 0 0 −203 0.00 6 3 Empty FALSE 166 240 0 0 476 0.00 6 4 Empty FALSE196 240 0 0 214 0.00 6 5 Empty FALSE 226 240 0 0 695 0.00 6 6 EmptyFALSE 257 240 0 0 218 0.00 6 7 Cy3 FALSE 287 240 1 3 1125959 9.57 6 8Empty FALSE 318 240 0 0 107 0.00 6 9 Empty FALSE 348 240 0 0 874 0.00 610 Empty FALSE 378 240 0 0 617 0.00 6 11 Empty FALSE 409 240 0 0 5280.00 6 12 Empty FALSE 439 240 0 0 580 0.00 7 1 Cy3 FALSE 105 271 1 −11734877 9.71 7 2 Empty FALSE 135 271 0 0 −634 0.00 7 3 Empty FALSE 166271 0 0 −69 0.00 7 4 Empty FALSE 196 271 0 0 276 0.00 7 5 Empty FALSE226 271 0 0 −199 0.00 7 6 Cy3 FALSE 257 271 0 −2 1581737 10.49 7 7 Cy3FALSE 287 271 0 0 1522026 9.37 7 8 Empty FALSE 318 271 0 0 −748 0.00 7 9Empty FALSE 348 271 0 0 2635 0.00 7 10 Empty FALSE 378 271 0 0 747 0.007 11 Empty FALSE 409 271 0 0 246 0.00 7 12 Cy3 FALSE 439 271 −1 01640104 10.37 8 1 Empty FALSE 105 301 0 0 586 0.00 8 2 Empty FALSE 135301 0 0 439 0.00 8 3 Probe 31 FALSE 166 301 1 −1 2008843 10.06 8 4 EmptyFALSE 196 301 0 0 247 0.00 8 5 Empty FALSE 226 301 0 0 319 0.00 8 6 HFALSE 257 301 −1 0 1534132 10.46 8 7 Empty FALSE 287 301 0 0 −477 0.00 88 Empty FALSE 318 301 0 0 13815 0.00 8 9 Probe 31 FALSE 348 301 −1 −11704828 10.04 8 10 Empty FALSE 378 301 0 0 260 0.00 8 11 Empty FALSE 409301 0 0 2993 0.00 8 12 H FALSE 439 301 0 2 1569671 9.96 9 1 Empty FALSE105 332 0 0 148 0.00 9 2 Probe 14 FALSE 135 332 2 −3 1969286 9.77 9 3Probe 35 FALSE 166 332 3 −1 1636381 9.53 9 4 Empty FALSE 196 332 0 0 7920.00 9 5 Empty FALSE 226 332 0 0 −377 0.00 9 6 Empty FALSE 257 332 0 0594 0.00 9 7 Empty FALSE 287 332 0 0 570 0.00 9 8 Probe 14 FALSE 318 3321 −3 1812677 10.36 9 9 Probe 35 FALSE 348 332 0 −2 1881764 9.97 9 10Empty FALSE 378 332 0 0 444 0.00 9 11 Empty FALSE 409 332 0 0 −423 0.009 12 Empty FALSE 439 332 0 0 287 0.00 10 1 Empty FALSE 105 362 0 0 4260.00 10 2 Empty FALSE 135 362 0 0 −23 0.00 10 3 Probe 36 FALSE 166 362 1−4 1582212 9.85 10 4 Empty FALSE 196 362 0 0 1518 0.00 10 5 Probe 29FALSE 226 362 1 −1 1629291 11.09 10 6 Empty FALSE 257 362 0 0 4511 0.0010 7 Empty FALSE 287 362 0 0 201 0.00 10 8 Empty FALSE 318 362 0 0 −3970.00 10 9 Probe 36 FALSE 348 362 −1 1 1683589 9.69 10 10 Empty FALSE 378362 0 0 75 0.00 10 11 Probe 29 FALSE 409 362 0 1 1763951 10.31 10 12Empty FALSE 439 362 0 0 607 0.00 11 1 Empty FALSE 105 392 0 0 −321 0.0011 2 Empty FALSE 135 392 0 0 437 0.00 11 3 Probe 37 FALSE 166 392 0 −41782130 9.95 11 4 dN20 FALSE 196 392 3 −2 1886735 10.28 11 5 Empty FALSE226 392 0 0 293 0.00 11 6 Probe 90 FALSE 257 392 0 −4 1569567 10.77 11 7Empty FALSE 287 392 0 0 1805 0.00 11 8 Empty FALSE 318 392 0 0 −262 0.0011 9 Probe 37 FALSE 348 392 −1 −3 1872819 9.83 11 10 dN20 FALSE 378 3921 −4 2034194 10.21 11 11 Empty FALSE 409 392 0 0 −258 0.00 11 12 Probe90 FALSE 439 392 0 0 1693534 10.95 12 1 Cy3 FALSE 105 423 3 −1 16584338.06 12 2 Empty FALSE 135 423 0 0 521 0.00 12 3 Empty FALSE 166 423 0 0285 0.00 12 4 Empty FALSE 196 423 0 0 −447 0.00 12 5 Empty FALSE 226 4230 0 −436 0.00 12 6 Empty FALSE 257 423 0 0 −129 0.00 12 7 Cy3 FALSE 287423 1 0 1392853 8.72 12 8 Empty FALSE 318 423 0 0 257 0.00 12 9 EmptyFALSE 348 423 0 0 649 0.00 12 10 Empty FALSE 378 423 0 0 −108 0.00 12 11Empty FALSE 409 423 0 0 64 0.00 12 12 Empty FALSE 439 423 0 0 −84 0.00

Algorithm 2

This process takes two pictures of the post-hybridization array: onewith normal exposure (Image_NormalExposure) and one with high-exposureto emphasize the Cy3 beacon (Image_HighExposure).

-   1. Read Image_HighExposure and Image_NormalExposure into memory.-   2. Read from QC text file.-   3. Operate on Image_HighExposure Image to find grid.-   a. Take the Cy5_HighExposure image, apply digital filtering and    pixel operation to produce an image with uniform and zero-valued    background.-   b. Threshold the image to into a binary image.-   c. Apply particle analysis to the binary image to identified,    filtered objects based on size. Measure and record parameters of the    objects: center of mass, bounding box, particle area and    ellipticity.-   d. Check to see if the number of objects identified in step 3C meets    the minimum requirement, otherwise reject the slide.-   e. Find grid, similar to step 6 in Algorithm 1.-   f. Rotate the image so the angle formed by the Cy3 drops is less    than 0.2 degree from the horizontal axis.-   g. Fine-tune the grid, similar to step 8 in Algorithm 1.-   4. Apply the grid found in Image_HighExposure to the    Image_NormalExposure.-   5. Using X-Deviation, Y-Deviation, diameter and reject flag from the    QC file to determine the relevant spot parameters.-   a. If Reject Flag is true, then exclude the spot from analysis.-   b. Spot X coordinate: X coordinate of the center of the grid PLUS    X-Deviation.-   c. Spot X coordinate: Y coordinate of the center of the grid PLUS    Y-Deviation.-   d. Spot diameter: Spot diameter from QC data.-   6. Calculate intensity of spot and background.-   7. Perform final calculation to determine analytical results.

Example 4

A protein microarray assembly is constructed using gel drop elementscontaining antibodies. Glass slides with printed gel element microarraysare blocked with PBS containing 1% BSA for 1 hour at room temperature.The slides are rinsed with DI water and allowed to air dry in adust-free environment. The microarray assembly is then assembled withthe blocked glass slide, laser cut 256M tape from Adchem, hydrophilicLexan film, and a reservoir. Approximately 0.5 mL of SEB (1 μg/mL in PBSwith 0.05% Tween-20 and 1% BSA) is pipetted into the reservoir of themicroarray system, and continuously imbibes through the array chamber atroom temperature. Next, 0.2 mL of anti-SEB monoclonal antibody dilutionin PBST with 1% BSA is pipetted into the reservoir of the microarraysystem, which continuously imbibes through the array chamber and intothe waste chamber. Then, 0.2 mL of PBST is pipetted into the reservoirof the microarray system, which continuously imbibes through the arraychamber and into the absorbent of the waste chamber. Subsequently, 0.1mL of Alexa 568 labeled anti-mouse antibody at 2 μg/mL in PBST with 1%BSA is pipetted into the reservoir of the microarray system, whichcontinuously imbibes through the array chamber and into the absorbent ofthe waste chamber. An additional 0.2 mL of PBST wash is pipetted intothe reservoir of the microarray system, which continuously imbibesthrough the array chamber and into the absorbent of the waste chamber.The microarray system is then imaged using a green laser (532 nm) with605 nm filter on Aurora PortArray 5000.

Example 5

Oligonucleotide mixtures are synthesized for MRSA according to the arraymap shown in FIG. 9. Each probe is synthesized along with the internalcontrol probe Cy5. Additional Cy3 control probes, attached to the sameoligonucleotide sequence, are also mixed with the Cy5 control probes.Cy3/Cy5 spots are printed in concentrations that range from 0.1 nM to 10μM in 1 log concentration changes for the purposes of establishing acalibration curve. An imaging system consists of two optical trains.Both optical trains consist of an LED and a non-cooled CCD camera. Oneoptical train is for detecting Cy3 spots (550 nm excitation and 570 nmemission), and the other optical train is for detecting Cy5 spots (650nm excitation and 670 nm emission). The optical trains are fixed inspace in relation to the instrument. A moving stage moves the array tothe green channel and 10 images are acquired to improve the dynamicrange; acquisition of multiple images at short exposure times preventssaturation that may occur as a result of using materials with highautofluorescence while also allowing signal averaging to reduce theeffect of random noise. The stage moves the array to the red channel and10 images are acquired. The process repeats 5 times to account forpossible misalignment due to positional accuracies, improper exposuretime, out of focus spots and/or any other anomalies that mightcompromise proper imaging. A calibration curve is plotted with respectto the Cy3/Cy5 serial dilution of concentrations as shown by the outerboundary of the array in the assembly map below. The calibration curve,derived from this concentration gradient, is intended to correct forfactors that affect the entire assembly such as shelf-life degradationof the probes, temperature, changes in UV dosage, synthesis variations,or any systemic artifact that can result in irreproducible behavior. Thecalibration curve for Cy5 is plotted during analysis with thecalibration curve for Cy3.

I _(red) =m _(red)×moles(Cy5)+b _(red)

I _(green) =m _(green)×moles(Cy3)+^(b)green

where I_(red) and I_(green) are background-subtracted integralintensities. The slopes, m_(red) and m_(green), and the intercepts,b_(red) and b_(green), are calculated from these calibration curves.Averages of the calibration curves are plotted and outliers arerejected. To account for irreproducibility from spot-to-spot,assembly-to-assembly and lot-to-lot, the Cy5 background-subtractedintegral intensity value is calculated for each spot. During synthesis,the probe (14, 31, 35, 36, 37, 29, 90, H, or dN20) concentrations foreach spot have an equimolar concentration of oligonucleotide probe andCy5 fluorophores for each spot. Thus, the following relationship holds:

Cy3 concentration≈Cy5 concentration.

Therefore,

I _(green),_(saturation) ≈m _(green)×moles(Cy5)+b _(green)

where I_(green,saturation) represents the hypothetical situation whereall probes in the gel element are bound to labeled-Cy3 molecules. Note,moles(Cy5) replaces moles(Cy3) because of equimolar equivalency in theequation above. The background-corrected integral intensity of theprobes for spots that determine presence of MRSA is measured at therepresentative spots, and calculated. If this intensity meets thefollowing criteria:

$\frac{I_{{green},{meas}}}{I_{{green},{saturation}}} > 0.001$

the value is considered positive. That is, more than 0.1% of thepossible probe molecules have bound target with Cy3 labels. This methodmay also be used for quantitation.

Example 6

Kiss-cut tape reels are manufactured for the spacer tape reel, whichcontains the spacer cut out, and the cover tape reel are pre-punchedwith the inlet and vent fill holes. As shown in FIG. 5, duringproduction, the substrate film reel unravels and the release liner iscollected on the top reel. The gel element printer prints the gelelements on the substrate film. The film is then exposed to UV under aninert atmosphere (e.g., Argon gas). Positive pressure of Argon is slowlyadded to the Argon chamber, and since its density is greater than air,it settles to the bottom of the chamber where the substrate is. Thisallows for a low flow rate of Argon into the chamber, and thus requiresminimal demand on room make-up air. To ensure that no unpolymerizedpolymer is left on the substrate, the substrate travels through a washstation, which is positioned in such a way as to eliminate any splashinto the polymerization chamber. The washed arrays are dried with aconventional air knife assembly. A QC camera ensures that that theelements are printed within specification and a rejection marker alertsthe operator to discard assemblies out of specification. Thedouble-sided spacer tape, which defines the microarray chamber, isunraveled and bonded to the substrate. The openings in the spacer tapeare designed to allow for fairly loose tolerances during the laminationof the spacer tape to the substrate, which allows the manufacture of gelelement arrays of variable geometry and complexity without modifying theassembly line. A rollable absorbent is unwound and mounted to the wastechamber of the assembly where it is sealed in place with either anadhesive or double-sided tape. An alternative strategy for including theabsorbent is to use a pick-and-place robot to insert the absorbent intoa waste chamber. The current flow cell design uses an additional spacerlayer to accommodate an absorbent that is twice as thick as the reactionchamber. Finally, the cover film, which has fill holes, is applied. Thefill holes can be considerably larger or smaller than the holes in thespacer allowing for loose tolerances during alignment. A guillotine thencuts the tape into the appropriate size.

Pin-printing robots typically feature a print head that is populatedwith precisely machined pins. The print head is attached to a precisionxyz-axis control aim (FIG. 7). The control arm is responsible for movingthe print head with micron-accuracy between the printing solution sourceplate (e.g., a 384 well microtiter plate), the substrate printingstation (the platen), and a wash station (for cleaning the pins betweendeposition of unique solutions). Alternatively, a high-throughput,non-contact print head deposits multiple solutions simultaneously withinthe microarray-PCR reaction chamber.

In some embodiments, the entire microarray is printed in asingle-stroke. Electrical Discharge Machining (EDM) can be used tocreate print heads with 125 micron pins (diameter of pins currentlyused) and the presently-used 300 micron centers. Additionally, ParallelSynthesis Designs offers 24576 well plates, which has wells with 560micron centers. This translates to approximately 350 spots per squarecm. While this many spots is sufficiently adequate for most diagnosticapplications, tests that require additional spots can be accommodated byarranging multiple printers serially in the assembly line, increasingprinting time only by the travel from one printer to the next. Anotherembodiment for printing on a moving film includes the use of anon-contact printer, which implements a piezoelectric crystal and acapillary that aspirates the microarray printing solution and dispensespicoliter to nanoliter drops onto the substrate. The printing head mayinclude multiple capillaries for simultaneously printing an array ofunique microarrays spots with distinct probes. Furthermore, this printhead may be a high density array of capillaries for increasing thenumber of unique microarray spots. Alternatively, the print head mayraster across and up and down the substrate film to print replicatespots or the print head raster method may be used to aspirate a separatepolymer-probe suspension for printing multiple unique spots using thesame print head. Another option is to have the substrate film re-loadedonto the reel-to-reel system following a first print pass, so as tore-print additional (unique) spots for each microarray on the roll ofsubstrate film. This re-printing approach may include fiducials toproperly align the film when printing on the second, third or n^(th)pass. One example of a relevant fiducial is the use of perforated edges,such as those used with 35 mm film. Another printing option includesacoustic ejection, a non-contact method available from Labcyte, in whichhigh frequency sound waves eject nL droplets from a source plate to adestination plate

Example 7

FIG. 8 shows the result of printing Cy3 gel elements on a 0.005″polyester film that was purchased from McMaster-Carr (Santa Fe Springs,Calif.) in a roll format. The film was placed in the vacuum chuck shownin FIG. 9 and printed on. It was imaged using bright field illuminationbefore and after polymerization. This array was also imaged with anAkonni imager that consists of an LED and a non-cooled camera.Subsequent to this printing, an MRSA array, described above, was printedon the polyester film and exposed to a Qiagen MasterMix using 300 pg ofpurified MRSA DNA. Thermocycling was performed on a Quanta Bioscienceslide block thermocycler. The result when using a rollable film for thetop and bottom surfaces as well as the spacer tape is shown in FIG. 10,which shows positive identification of MRSA. FIG. 11 shows areel-to-reel printing setup with a BioDot Ultranon-contact array printer(top panel) and video frames of non-contact printing using the BioDotUltra on a moving film that has not been chemically treated or modified(bottom panels). These results demonstrate the feasibility of printingmicroarrays on a moving film with a non-contact array printer, whichallows for high speed and low cost production of microarray assembliesof the present application.

Example 8

FIGS. 12-14 show the array image analysis process. FIG. 12 is an imageof an array taken at factory QC with red channel imager to extract arrayQC data. The QC software algorithm automatically finds each spot in thearray and associates each spot to its perspective location in the arraygrid. The software displays the 12 by 12 square grid and draws circlearound each drop. The QC software calculates the relative location(Deviation X, Deviation Y) of each circle within its bounding squaregrid box and stores the information as part of the QC data to assist inpost-hybridization image analysis.

FIG. 13 is an image of the array is taken after hybridization withend-user's green channel imager. The image analysis software locates thearray grid based on Cy3 fluorescent beacons and draws the 12 by 12square grid. The image analysis then use the relative location of thespot (Deviation X and Deviation Y from the array QC file) to locate eachspot and draws circle around the spot for visual indication. Note thatwith the assistance of array QC data, the software was able to locateevery spot on the arrays and draw circles that fully enclose each spot.

FIG. 14 is an image of the array is taken after hybridization withend-user's green channel imager. The image analysis software locates thearray grid based on Cy3 fluorescent beacons and draws the 12 by 12square grid. For this image, image analysis software does not haveaccess to information on relative spot location from the array QC file.Instead, the image analysis software identified each spot by assume thecenter of each square grid cell is the center of the spot. The softwarehas incorrectly identified several spots, by drawing circles that do notfully enclose the spot. The incorrectly identified spots include spotslocated in: row 2 column 2, row 11 column 6. Note this method of imageanalysis without array QC data is less robust in spot identificationthan image analysis with array QC data shown in FIG. 13.

1-25. (canceled)
 26. A method of detecting a target molecule in asample, comprising: loading the sample in an microarray assembly,wherein the microarray assembly comprises: a funnel-shaped array chamberwith a sample inlet at a first end located at the start of the funnelshape, a sample outlet at a second end located at the end of the funnelshape, a top interior surface, a bottom interior surface, side walls anda microarray located on the bottom interior surface; and a waste chamberthat is in fluid communication with the outlet of the array chamber,wherein the array chamber comprises a hydrophilic interior surfacepositioned to facilitate complete filling of the array chamber by awater-based fluid and the continuous flow of the fluid from the sampleinlet to the sample outlet, wherein the cross-sectional area at thefirst end of the array chamber is larger than the cross-sectional areaat the second end of the array chamber, and wherein the array chamber isin the shape of a channel and wherein the cross-sectional area of thearray chamber decreases in a continuous manner from the first end to thesecond end such that the capillary pressure continuously increase as thewater-based fluid in the array chamber approached the waste chamber andtherefore provides continuous wicking of the water-based fluid in thearray chamber in the direction of the waste chamber; incubating thesample in the array chamber to allow binding of the target molecule tothe microarray; and detecting the target molecule bound to themicroarray.
 27. The method of claim 35, wherein the cross-sectional areaof the array chamber at the first end is three-times larger than thecross-sectional area of the array chamber at the second.
 28. The methodof claim 35, wherein the microarray comprises a plurality of array spotsarranged in a single row extending from the first end to the second endof the array chamber.
 29. The method of claim 35, wherein the microarraycomprises a plurality of parallel array strips that are perpendicular tothe direction of sample flow in the array chamber.
 30. The method ofclaim 35, wherein the microarray is a gel spot microarray.
 31. Themethod of claim 39, wherein the gel spot microarray is an antibodyarray.
 32. The method of claim 39, wherein the gel spot microarray is aprotein array.
 33. The method of claim 39, wherein gel spots of the gelspot microarray comprise protein probes covalently cross-linked topolymer backbone of the gel spots.
 34. The method of claim 39, whereinthe microarray is an antibody array.
 35. The method of claim 35, whereinmicroarray assembly comprises a substrate layer, a cover layer and aspacer layer located between the substrate layer and the cover layer.36. The method of claim 44, wherein the spacer layer is a double-sidedtape and wherein the array chamber is formed within the spacer layer.37. The method of claim 35, wherein the microarray assembly comprises asubstrate layer and a cover layer, wherein the substrate layer is aninjection molded plastic with features that create walls of the arraychamber and a pocket for the waste chamber.
 38. The method of claim 47,wherein the cover, layer is a hydrophilic film laminated to thesubstrate layer.
 39. The method of claim 35, further comprisingamplifying the target molecule in the array chamber.
 40. The method ofclaim 48, wherein the target molecule is amplified by polymerase chainreaction.